The polymerase chain reaction (PCR) is a sensitive DNA amplification procedure that permits the selection and detection of specific nucleic acids from a complex mixture. In its most rudimentary form, PCR is employed using a sample that contains a target nucleic acid (DNA), a set of DNA primers that hybridize to the target, and a DNA polymerase that is capable of primer-based synthesis of complementary strands of the target. During the nucleic acid amplification process, the target:primer:polymerase mixture is subjected to successive rounds of heating at different temperatures to facilitate target DNA strand separation (performed at ˜90-99° C.), primer:target DNA strand annealing (performed at ˜40-70° C.), and DNA polymerase-mediated primer elongation (performed at ˜50-72° C.) to create new complementary target strands. Because the reaction may be subjected to ˜25-45 rounds of cycling to yield the desired DNA amplification product, PCR is usually conducted using thermal stable DNA polymerases that can withstand the very high temperatures associated with target strand separation without suffering inactivation due to heat-induced protein denaturation. Since its introduction in the mid-1980's, PCR has become the de facto standard for detecting minute quantities of nucleic acids in samples, and obtaining specific genes from complex DNA genomes and samples.
A major problem with diagnostic and forensic techniques based on PCR is the false-negative reactions or low sensitivity caused by inhibitory substances that interfere with PCR (1, 2, 3). Of particular clinical importance is the PCR analysis of blood samples, which represents the largest fraction of human health related tests for diagnosis of genetic diseases, virus and microbial infections, blood typing, and safe blood banking. Various studies indicate that the inhibitory effect of blood on PCR is primarily associated with direct inactivation of the thermostable DNA polymerase and/or capturing or degradation of the target DNA and primers. It has been reported that the protease activity in blood also contributes to the reduced efficiency of PCR (1-5, 7, 10, 12).
The blood resistance characteristics of the thermostable DNA polymerases vary with the source of the enzyme (6). Widely used thermostable polymerases like Thermus aquaticus DNA polymerase (Taq) and AmpliTaq Gold® are completely inhibited in the presence of 0.004-0.2% whole human blood (vol/vol; 3, 4, 6). Various agents have been tested for reducing the inhibitory effect of blood on Taq. It was found that an addition of betaine, bovine serum albumin, the single-stranded DNA binding protein of the T4 32 gene (gp 32), or a cocktail of protease inhibitors can partially relieve the blood inhibition and allow Taq to work in up to 2% blood (vol/vol), although this effect could be sample specific (3, 8, 9, 11).
Several major inhibitors of PCR in human blood have been characterized such as immunoglobulin G, hemoglobin, lactoferrin and excess of leukocyte DNA (4, 7, 10). The IgG, hemoglobin, and lactoferin have been purified from plasma, erythrocytes and leukocytes, respectively, using size-exclusion and anion-exchange chromatography (4, 7). The heme has been reported to inactivate the Taq polymerase by binding to its catalytic domain (10), while the mechanism of action of the other inhibitory components is more poorly understood. The inhibitory effect of IgG can be reduced when this plasma fraction is heated at 95° C. before adding it to PCR, or with the addition of excess non-target DNA to the PCR mixture. However, heating of IgG together with target DNA at 95° C. was found to block amplification. Inhibition by IgG may be due to an interaction with the single-stranded DNA fraction in the target DNA. The inhibitory effect could be removed also by treating the plasma with DNA-agarose beads prior to amplification (4).
Other complicating factors include EDTA and heparin, used as anti-coagulants, which can also inhibit DNA amplification. The addition of heparinase has been shown to counteract the heparin-mediated inhibition (13, 14). Therefore, various laboratory procedures of sample preparation have been developed to reduce the inhibitory effect of blood. The DNA purification methods suitable for PCR can include additional steps like dialysis, treatment with DNA-agarose beads or Chelex 100 resin, multiple DNA washes, or a combination of dilution with buffer which causes lysis of red blood cells, centrifugation to recover the white blood cells, washing with NaOH and the addition of bovine serum albumin (2, 3, 15-19).
These pre-treatment steps of the blood samples are generally time-consuming, labor-intensive, and can be sample specific. The guanidinium thiocianate method for DNA isolation is not suitable for reliable detection of Mycobacterium tuberculosis in clinical samples. An alternative method of DNA purification with protease K treatment followed by phenol-chloroform extraction has to be employed to relieve the inhibition (20). Separation with a QIAamp kit followed by dialysis with a Millipore filter are required for eliminating the heme inhibition of hepatitis B virus detection (21). In addition, some the above steps carry a risk of target DNA losses and are not suitable for automation. Moreover, even commercial kits specially formulated for DNA purification from blood samples such as QIAmp or GeneReleaser are not always satisfactory. The reason is due to an incomplete removal of Taq inhibitors, which can result in false-negative results. For example, 14% of the human blood samples tested for hepatitis B virus yielded false-negative results when using such blood kits (21).
The objective of achieving specificity of amplification reactions for samples containing whole blood is further complicated by two types of unwanted DNA synthesis reactions that occur during PCR. Both types of side-reactions are frequently competitive with the desired target and can lead to impure product or failed amplification. This is particularly problematic for PCR assays containing a low copy number of the nucleic acid template target, wherein the PCR conditions are modified to include a greater number of amplification cycles to achieve an adequate yield of the desired amplification product.
The first type of unwanted DNA synthesis is priming on less specific sequences in the template. This is only an issue if the template is contaminated with single-stranded nucleic acid or if the template is single-stranded, which is the case if the DNA preparation has been subjected to melting conditions during its isolation.
The second type of unwanted DNA synthesis is primers acting as templates for themselves and/or each other, with at least the result of modifying their 3′ ends by the addition of additional nucleotides. These so-modified primers are able to anneal to the nucleic acid target; however, they do not serve as primers for complementary strand synthesis due to the presence of mismatched nucleotides at the site of elongation between the 3′ end of the primer and the desired target. This problem is often referred to as “primer dimer”, although this name is not accurately descriptive. This problem can often be reduced or avoided by careful primer design, and it is more of a problem with multiplex PCR, since there is more opportunity for accidental homology among multiple pairs of primers.
A procedure known as “hot start PCR” avoids the occurrence of both types of unwanted DNA synthesis side-reactions. According to this method, the enzyme DNA polymerase, or a buffer component essential to its activity, such as the magnesium (II) cation and/or the dNTPs, is withheld from the other PCR assay mixture ingredients until the PCR reaction has been heated to at least the normal primer-annealing (or, preferably, the DNA extension) temperature (55-75° C., optimal 68° C.). At this temperature the primers can presumably not form stable duplexes with themselves or at unwanted template sequences. After the selective temperature is achieved, the omitted component is added to reaction to reconstitute a functional amplification mixture.
Typical hot start PCR procedures are not only labor-intensive, they expose the PCR reactions to contamination with each other and with molecules that have been previously amplified in the thermal cycler machine.
The more standard ways of executing a hot start consist of formulating the PCR reaction in two parts, such that the DNA polymerase is not able to act on the DNA until the two portions are combined at high temperature, usually 65-85° C. For instance, an initial solution containing all of the magnesium is introduced to the reaction tube encapsulated in a wax bead or sealed under a layer of wax. The rest of the reaction, without Mg, is then added, along with an overlay of oil, if appropriate. While the reaction heats for the first cycle, the wax melts and floats to the surface, allowing the magnesium to mix with the reaction volume. The DNA polymerase activity is therefore reconstituted at a temperature that does not allow non-specific or unwanted primer interactions. A great drawback to the wax method comes after the PCR cycling is complete, and the product must be withdrawn for analysis. The wax then tends to plug the pipette tip, greatly adding to the time and effort of reaction analysis.
Recently, a method of hot start which is not hot at all, but which uses anti-Taq antibodies, has been described, patented and made commercially available (33-35). The antibodies largely neutralize the enzyme activity of the Taq polymerase, and can be added any time prior to the primers, or be conveniently present during storage of the stock enzyme. The antibodies are thermolabile, thus permitting the Taq polymerase to resume activity after the first heat step. The antibodies so far developed for this method must be used in 10-fold molar excess and are expensive. Furthermore, the antibodies inhibit some long PCR assays that are conducted with the KlentaqLA polymerase mixture.
A chemically inactivated form of the Taq polymerase has been introduced recently, termed AmpliTaq Gold®. The nature of the inactivation is proprietary, but the inactivation is reversible by heating the polymerase at 95° C. This method may be even more convenient than the other methods, but it has at least one current disadvantage: the time for reactivation is about 10 minutes at 95° C. This procedure is incompatible with long PCR applications, as this treatment would excessively depurinate nucleic acid targets longer than a few kb.
Thus, the analysis of whole blood samples using PCR would be benefited by the discovery of new reagents and methods that overcome the aforementioned shortcomings of current PCR technologies. The invention disclosed herein addresses and solves many of these shortcomings.